Recent reports on food-borne illness clearly indicate the economic and public health significance of salmonellosis and campylobacteriosis. In 1989, the number of confirmed salmonella cases in England and Wales rose to 29,998 (Cooke, M E. (1990). The Lancet 336:790-793.) while those of campylobacter rose to 32,359 (Skitrow M B.(a) (1990). Proceedings of the 14th International Symposium of the ICFMH. Telemark, Norway; (b) (1990). The Lancet 336: 921-23.) with estimated average tangible costs per case of .English Pound.2,240 (Yule, B F et al (1988) Epidemiology and Infection 100: 35-42.) and .English Pound.273 (Skitrow 1990 (a) (b)) respectively. Similar data exist for the U.S.A. where approximately 40,000 salmonella cases are reported annually, with average hospitalization or treatment costs rising up to $4,350 per case (Roberts T. (1988). Poultry Science 67: 936-43.). The incidence of campylobacter is also high and, for example, in the state of Washington, has been estimated at 100,000/150,000 (Todd, E. (1990). The Lancet 336: 788-90.). Confirmed cases of disease probably represent only 1 to 10% of the total number of clinically significant cases (Aserkoff B et al (1970). American Journal of Epidemiology 92: 13-24.; Oosterom (1990) Procedings of the 14th International Symposium of the ICFMH. Telemark, Norway.; Skirrow 1990a, b).
Following the compulsory heat treatment of milk in 1983 poultry meat has become the most incriminated food vehicle of salmonellosis and campylobacteriosis in the UK. The Public Health Laboratory Service ((1989) PHLS Microbiology Digest 6: 1-9) found that 60 to 80% of retail chickens in the UK were contaminated with salmonella while reports from other countries indicate levels ranging between 5 and 73%. The incidence of campylobacter may be even higher, and in some studies all chicken carcasses examined were contaminated (Hood, A M et al (1988) Epidemiology and Infection 100: 17-25.; Lammerding, A M et al (1988). Journal of Food Protection 51: 47-52.). In addition, contamination of red meats leads to sporadic cases of both salmonella and campylobacter disease. Thus there is considerable pressure on the meat and poultry industries to improve the bacteriological quality of their products by developing and applying decontamination processes.
Considerable effort has been devoted to the development of chemical decontamination techniques. However, although a large number of chemical treatments have been tested (Table 1), these have in general proven either unsuccessful on application, or have had adverse effects on the appearance, odour or taste of meat, occasionally leaving undesirable residues. Chlorine is the only chemical currently in use in poultry processing operations and maximum levels of 20 ppm in the spray wash are recommended by the EC, although higher concentrations (40 ppm) may be required to reduce bacterial populations in both carcasses and equipment. Chlorine, however, can damage processing equipment and leads to the formation of potential carcinogens such as chlorinated hydrocarbons when contacted with organic matter.
There has been for many years a considerable interest in using the enzyme lysozyme as a food preservative. Lysozyme is a naturally occurring antimicrobial agent, has no adverse effects on man and is present, for example, in tears and milk. It can also easily be recovered through industrial processes from egg-white and is approved for food use in Europe, Japan and the U.S.A. (Hughey, V L et al (1987). Applied and Environmental Microbiology 53: 2165-2170). Table 2 lists the variety of food products that may be preserved by treatments involving lysozyme derived from milk or egg-white.
Lysozyme may cause rapid lysis of Gram-positive bacteria but unless subjected to modifying treatments, cells of Gram-negative bacteria are resistant. Lysozyme hydrolyses peptidoglycan, a polymer present in the cell walls of Gram-positive and Gram-negative bacteria which maintains rigidity of the wall. In Gram-positive organisms, peptidoglycan is present throughout the cell wall, which consists of a more or less homogeneous matrix of peptidoglycan and other polymers.
However, in Gram-negative bacteria, peptidoglycan exists as a discrete layer which is protected from the environment by a lipid outer membrane which acts as a permeability barrier against large molecules, such as lysozyme (MW 14,900 D). Thus, in the absence of procedures for modifying the outer membrane, only foods dominated by a Gram-positive bacterial flora may be preserved by lysozyme.
The outer membrane of Gram-negative bacteria may be disrupted by heat (Becker M E et al (1954) Archives of Biochemistry and Biophysics 53: 402-410; freezing and thawing (Kohn, N R. (1960) Journal of Bacteriology 79: 697-706.); extraction of the lipopolysaccharide component of the outer membrane with lipid solvents or alkali (Becker et al (1955) Archives of Biochemistry and Biophysics 55: 257-269.), starvation at extreme pH environments (Nakamura, O. (1923) Immunitatsforschrift 38: 425-449; Grula, E A et al (1957) Canadian Journal of Microbiology 3: 13-21), treatments with EDTA (Repaske, R. (1956) Biochemica et Biophysica Acta 22: 189-191 and (1958) Biochemica et Biophysica Acta 30: 225-232), detergents (Colobert L. (1957) Comptes Rendues 245: 1674-1676.), or polybasic antibiotics (Warren G H (1957) Journal of Bacteriology 74: 788-793.). Hypo-osmotic shock in the presence of lysozyme (Birdsell, D C et al 1967. Journal of Bacteriology 93: 427-437; Witholt, B H et al (1976) Biochimica et Biophysica Acta 443: 534-44.) has also been demonstrated to kill Escherichia coli (E. coli) cells suspended in Tris-EDTA buffer and plasmolysed by the addition of sucrose.
Procedures involving EDTA and lysozyme have been tested on shrimp (Chandler R et al (1980) Applied Microbiology and Biotechnology 10: 253-258.) and poultry (see Table 2), but although some reduction in contamination levels was observed the use of EDTA makes the technique generally inapplicable to food-treatment. Osmotic shock procedures (Withholt B H et al 1976) might also be acceptable in food processing if the requirement for EDTA could be eliminated.
The transfer of bacteria from typical growth media (a.sub.w 0.999) to media made hypertonic by the addition of solutes which do not penetrate cells, such as sucrose or NaCl, is accompanied by an abrupt loss of cell water. Gram-negative bacteria subjected to such hyper-osmotic shock undergo "plasmolysis" which is characterised by loss of turgor pressure, shrinkage of the protoplast (Witter L (1987) Vol. 1: 1-35. In T J Montville (ed), Food Microbiology. CRC Press, Florida.), retraction of the cytoplasmic membrane from the outer membrane (Scheie, P O. (1969) Journal of Bacteriology 98: 335-40.), or contraction of the whole cell (Alemohammad M M et al (1974) Journal of General Microbiology 82: 125-142.). Subsequent survival, growth rate and maximum population density then depends upon the a.sub.w of the medium and the rate and extent to which the osmoregulatory mechanisms (Booth, I R, et al (1988) Journal of Applied Bacteriology Symposium Supplement PP. 35-49; Csonka L N. (1989) Microbiological Reviews 53: 121-147) of the organism may be restored to regain cell water (Dhavises, G et al (1979) Microbios Letters 7: 105-115. and (1979) Microbios Letters 7: 149-59.).
Water uptake is achieved by `deplasmolysis`, which in contrast to plasmolysis requires the presence of an energy source in the medium and is characterised by uptake and accumulation of K+ ions and uptake and/or synthesis of certain organic osmolytes, referred to as compatible solutes or osmoprotectants.
In contrast, transfer of cells from media of low to high a.sub.w (water activity), thus effecting hypo-osmotic shock, results in an instantaneous influx of water and a concomitant increase in the cytoplasmic volume. However, cell volume increase in bacteria is generally limited by the presence of the cell wall which is relatively rigid and may withstand pressures of up to 100 atmospheres.
Although hypo-osmotic shock does not generally result in cell lysis, it may cause membrane disruption which can be demonstrated by the loss of intracellular solutes, such as ions, neutral and anionic sugars and phosphate esters (Leder, I G (1972) Journal of Bacteriology 111: 11-19; Tsapis A et al (1976) Biochimica et Biophysica Acta 469: 1-12.). Such loss has been described at optimum growth temperatures (30.degree.-37.degree. C.) and at 45.degree. C., as well as in combination with cold shock.
Cold shocks are achieved by rapidly lowering the temperature of cell suspensions, for example from 37.degree. C. to 0.degree. C. (Sherman, J M et al (1923) Journal of Bacteriology 8: 127-139.). The shock may result in cell death and cells from the exponential phase of growth are most susceptible (Jay, J. (1986) Modern Food Microbiology. 3rd ed Van Nostrand Reinhold Co Inc, NY.). Lysozyme has been reported to enhance lysis of exponential phase E. coli cells suspended in Tris-HCl buffer and subjected to cold shock (Scheie, P O. (1982) Biochimica et Biophysica Acta 716: 420-23.), though Tris-HCl may itself aid lysis of Gram-negative cells (Schindler, H et al (1979) American Chemical Society 18: 4425-30.).